Bottom Line:
In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period.Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue.This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

ABSTRACTCa(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

Fig4: Ca2+ sensitivity of FP-Aeq variants obtained in live cells. a Time course of bioluminescence of HEK-293 cells expressing the chimeras. Cells were incubated with CLZ-f, placed on the microscope stage, and stimulated with carbachol (50 μM) and later permeabilized with digitonin (30 μM) in the presence of Ca2+ to release all remaining counts (representative experiments). The left Y-axis shows the fractional rate of luminescence (L/Lmax) and the right Y-axis shows the percentage of counts remaining as a function of time. Different L/Lmax scales were used to reveal the Ca2+ oscillations, which prevented showing the full digitonin response. Images were acquired at 3 s per frame, and the intensity was averaged in single cells. b Cells expressing the indicated chimeric protein were incubated with CLZ-native, CLZ-f, or CLZ-hcp, exposed to carbachol and later permeabilized with digitonin/Ca2+, as shown in a. Log (L/Lmax) values correspond to the peak of the first Ca2+ response obtained with carbachol. The average of 11–42 cells from three to six independent experiments is shown for each variant

Mentions:
We also devised a protocol for estimating the Ca2+ sensitivity of the chimeras in live mammalian cells: a stimulus raising cytosolic Ca2+ to about 1 μM was followed by Ca2+ saturation of the probe; the fractional light intensity at 1 μM Ca2+ was an indication of the sensitivity of the probe. HEK-293 cells expressing the chimeras were reconstituted with CLZ-f, imaged, and stimulated with the muscarinic receptor agonist carbachol, followed by release of all remaining counts with digitonin and high Ca2+. Then, the fractional rate of luminescence (L/Lmax), which is proportional to Ca2+ concentration, was calculated in each cell over time (Fig. 4a).Fig. 4

Fig4: Ca2+ sensitivity of FP-Aeq variants obtained in live cells. a Time course of bioluminescence of HEK-293 cells expressing the chimeras. Cells were incubated with CLZ-f, placed on the microscope stage, and stimulated with carbachol (50 μM) and later permeabilized with digitonin (30 μM) in the presence of Ca2+ to release all remaining counts (representative experiments). The left Y-axis shows the fractional rate of luminescence (L/Lmax) and the right Y-axis shows the percentage of counts remaining as a function of time. Different L/Lmax scales were used to reveal the Ca2+ oscillations, which prevented showing the full digitonin response. Images were acquired at 3 s per frame, and the intensity was averaged in single cells. b Cells expressing the indicated chimeric protein were incubated with CLZ-native, CLZ-f, or CLZ-hcp, exposed to carbachol and later permeabilized with digitonin/Ca2+, as shown in a. Log (L/Lmax) values correspond to the peak of the first Ca2+ response obtained with carbachol. The average of 11–42 cells from three to six independent experiments is shown for each variant

Mentions:
We also devised a protocol for estimating the Ca2+ sensitivity of the chimeras in live mammalian cells: a stimulus raising cytosolic Ca2+ to about 1 μM was followed by Ca2+ saturation of the probe; the fractional light intensity at 1 μM Ca2+ was an indication of the sensitivity of the probe. HEK-293 cells expressing the chimeras were reconstituted with CLZ-f, imaged, and stimulated with the muscarinic receptor agonist carbachol, followed by release of all remaining counts with digitonin and high Ca2+. Then, the fractional rate of luminescence (L/Lmax), which is proportional to Ca2+ concentration, was calculated in each cell over time (Fig. 4a).Fig. 4

Bottom Line:
In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period.Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue.This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

ABSTRACTCa(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.